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1.  CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice 
PLoS ONE  2014;9(1):e86348.
Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.
doi:10.1371/journal.pone.0086348
PMCID: PMC3897723  PMID: 24466045
2.  TREM-1 Deficiency Can Attenuate Disease Severity without Affecting Pathogen Clearance 
PLoS Pathogens  2014;10(1):e1003900.
Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1−/− mice are viable, fertile and show no altered hematopoietic compartment. In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Trem1−/− mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1−/− mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1−/− mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1−/− mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1+/+ controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.
Author Summary
Triggering receptor expressed on myeloid cells-1 (TREM-1) is an immune receptor expressed by myeloid cells that has the capacity to augment pro-inflammatory responses in the context of a microbial infection. While a TREM-1-amplified response likely serves the efficient clearance of pathogens, it also bears the potential to cause substantial tissue damage or even death. Hence, TREM-1 appears a possible therapeutic target for tempering deleterious host-pathogen interactions. However, in models of bacterial sepsis controversial findings have been obtained regarding the requirement of TREM-1 for bacterial control - depending on the overall degree of the TREM-1 blockade that was achieved. In order to conclusively investigate harmful versus essential functions of TREM-1 in vivo, we have generated mice deficient in Trem1. Trem1−/− mice were subjected to experimentally-induced intestinal inflammation (as a model of a non-infectious, yet microbial-driven disease) and also analysed following infections with Leishmania major, influenza virus and Legionella pneumophila. Across all models analysed, Trem1−/− mice showed substantially reduced immune-associated disease. We thus describe a previously unanticipated pathogenic role for TREM-1 also during a parasitic and viral infection. Importantly, our data suggest that in certain diseases microbial control can be achieved in the context of blunted inflammation in the absence of TREM-1.
doi:10.1371/journal.ppat.1003900
PMCID: PMC3894224  PMID: 24453980
3.  Macrophage and T Cell Produced IL-10 Promotes Viral Chronicity 
PLoS Pathogens  2013;9(11):e1003735.
Chronic viral infections lead to CD8+ T cell exhaustion, characterized by impaired cytokine secretion. Presence of the immune-regulatory cytokine IL-10 promotes chronicity of Lymphocytic Choriomeningitis Virus (LCMV) Clone 13 infection, while absence of IL-10/IL-10R signaling early during infection results in viral clearance and higher percentages and numbers of antiviral, cytokine producing T cells. IL-10 is produced by several cell types during LCMV infection but it is currently unclear which cellular sources are responsible for induction of viral chronicity. Here, we demonstrate that although dendritic cells produce IL-10 and overall IL-10 mRNA levels decrease significantly in absence of CD11c+ cells, absence of IL-10 produced by CD11c+ cells failed to improve the LCMV-specific T cell response and control of LCMV infection. Similarly, NK cell specific IL-10 deficiency had no positive impact on the LCMV-specific T cell response or viral control, even though high percentages of NK cells produced IL-10 at early time points after infection. Interestingly, we found markedly improved T cell responses and clearance of normally chronic LCMV Clone 13 infection when either myeloid cells or T cells lacked IL-10 production and mice depleted of monocytes/macrophages or CD4+ T cells exhibited reduced overall levels of IL-10 mRNA. These data suggest that the decision whether LCMV infection becomes chronic or can be cleared critically depends on early CD4+ T cell and monocyte/macrophage produced IL-10.
Author Summary
Chronic viral infections like Hepatitis B and C Virus (HBV and HCV) and Human Immunodeficiency Virus (HIV) in humans affect more than 500 million people worldwide. While a robust T cell response is a hallmark of many acute infections one hurdle inhibiting the clearance of chronic viral infections is that the immune-suppressive cytokine IL-10 modulates the virus-host balance towards induction of T cell dysfunction. IL-10 is produced by several cell types during chronic Lymphocytic Choriomeningitis Virus (LCMV) infection but it is currently unclear which cellular sources are responsible to promote viral chronicity. Here, we demonstrate that T cell responses improved markedly, and that normally chronic LCMV Clone 13 infection could be cleared when either myeloid cells or T cells lacked IL-10 production. Furthermore, mice depleted of monocytes/macrophages or CD4+ T cells exhibited reduced overall levels of IL-10 mRNA. These data suggest that the decision whether LCMV infection becomes chronic or can be cleared critically depends on CD4+ T cell and monocyte/macrophage produced IL-10 early during the establishment of viral chronicity.
doi:10.1371/journal.ppat.1003735
PMCID: PMC3820745  PMID: 24244162
4.  Comparing the Kinetics of NK Cells, CD4, and CD8 T Cells in Murine Cytomegalovirus Infection 
NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response tomurine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H+ NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H+ NK cells, may be not be as robust as the secondary response observed in T cells.
doi:10.4049/jimmunol.1100416
PMCID: PMC3771860  PMID: 21697462
5.  Programmed death 1 protects from fatal circulatory failure during systemic virus infection of mice 
The Journal of Experimental Medicine  2012;209(13):2485-2499.
The PD-1–PD-L1 pathway inhibits perforin-mediated killing of PD-L1+ vascular endothelial cells by CD8+ T cells, thereby limiting vascular damage during systemic LCMV infection.
The inhibitory programmed death 1 (PD-1)–programmed death ligand 1 (PD-L1) pathway contributes to the functional down-regulation of T cell responses during persistent systemic and local virus infections. The blockade of PD-1–PD-L1–mediated inhibition is considered as a therapeutic approach to reinvigorate antiviral T cell responses. Yet previous studies reported that PD-L1–deficient mice develop fatal pathology during early systemic lymphocytic choriomeningitis virus (LCMV) infection, suggesting a host protective role of T cell down-regulation. As the exact mechanisms of pathology development remained unclear, we set out to delineate in detail the underlying pathogenesis. Mice deficient in PD-1–PD-L1 signaling or lacking PD-1 signaling in CD8 T cells succumbed to fatal CD8 T cell–mediated immunopathology early after systemic LCMV infection. In the absence of regulation via PD-1, CD8 T cells killed infected vascular endothelial cells via perforin-mediated cytolysis, thereby severely compromising vascular integrity. This resulted in systemic vascular leakage and a consequential collapse of the circulatory system. Our results indicate that the PD-1–PD-L1 pathway protects the vascular system from severe CD8 T cell–mediated damage during early systemic LCMV infection, highlighting a pivotal physiological role of T cell down-regulation and suggesting the potential development of immunopathological side effects when interfering with the PD-1–PD-L1 pathway during systemic virus infections.
doi:10.1084/jem.20121015
PMCID: PMC3526355  PMID: 23230000
6.  IL-21 Restricts Virus-driven Treg Cell Expansion in Chronic LCMV Infection 
PLoS Pathogens  2013;9(5):e1003362.
Foxp3+ regulatory T (Treg) cells are essential for the maintenance of immune homeostasis and tolerance. During viral infections, Treg cells can limit the immunopathology resulting from excessive inflammation, yet potentially inhibit effective antiviral T cell responses and promote virus persistence. We report here that the fast-replicating LCMV strain Docile triggers a massive expansion of the Treg population that directly correlates with the size of the virus inoculum and its tendency to establish a chronic, persistent infection. This Treg cell proliferation was greatly enhanced in IL-21R−/− mice and depletion of Treg cells partially rescued defective CD8+ T cell cytokine responses and improved viral clearance in some but not all organs. Notably, IL-21 inhibited Treg cell expansion in a cell intrinsic manner. Moreover, experimental augmentation of Treg cells driven by injection of IL-2/anti-IL-2 immune complexes drastically impaired the functionality of the antiviral T cell response and impeded virus clearance. As a consequence, mice became highly susceptible to chronic infection following exposure to low virus doses. These findings reveal virus-driven Treg cell proliferation as potential evasion strategy that facilitates T cell exhaustion and virus persistence. Furthermore, they suggest that besides its primary function as a direct survival signal for antiviral CD8+ T cells during chronic infections, IL-21 may also indirectly promote CD8+ T cell poly-functionality by restricting the suppressive activity of infection-induced Treg cells.
Author Summary
T cell exhaustion represents a state of T cell dysfunction associated with clinically relevant diseases, such as persistent viral infections or cancer. Although the molecular signature of exhausted T cells has been characterized in detail at the functional and transcriptional level, the immunological mechanisms that lead to T cell exhaustion during chronic infections remain poorly understood. Our present study reports two major findings that illustrate a pathway that contributes to T cell exhaustion during viral infection, and indicate its modulation by both, the pathogen and the host. First, we show that a persistence-inducing virus triggers the massive proliferation of Foxp3+ regulatory T (Treg) cells and demonstrate the potential of Treg cells to promote T cell exhaustion and chronic infection. Second, we identify IL-21 as a crucial host factor that antagonizes this virus-driven expansion of the Treg population in a cell intrinsic manner independent of IL-2. Thus, in addition to its known pre-dominant direct positive effects on antiviral T cells, IL-21 can also alleviate the suppressive activity of Treg cells. Together, these results suggest enhanced Treg cell responses as a mechanism of immune evasion that could be therapeutically targeted with IL-21.
doi:10.1371/journal.ppat.1003362
PMCID: PMC3656089  PMID: 23696736
7.  Impact of antigen specificity on CD4+ T cell activation in chronic HIV-1 infection 
BMC Infectious Diseases  2013;13:100.
Background
HIV infection induces chronic immune activation which is associated with accelerated disease progression; the causes of this activation, however, are incompletely understood. We investigated the activation status of CD4+ T cells specific for chronic herpes viruses and the non-persistent antigen tetanus toxoid (TT) in HIV positive and HIV negative donors to assess whether persistent infections contribute to chronic CD4+ T cell activation.
Methods
Untreated HIV+ patients and healthy, aged matched controls were recruited and activation levels assessed and compared between cells specific for persistent and non-persistent antigens. Activation levels on antigen-specific CD4+ T cells were measured by intracellular cytokine staining following in vitro stimulation with various recall antigens (CMV, EBV, HSV, VZV and TT) in conjunction with cell surface phenotyping.
Results
Activation levels of herpes virus-specific CD4+ T cell populations, assessed by co-expression of CD38 and HLA-DR, were significantly elevated in HIV+ individuals compared to normal controls and compared to TT-specific responses. In contrast, we found similar levels of activation of TT-specific CD4+ T cells in HIV+ and HIV- donors.
Conclusions
These results show a disparate distribution of immune activation within CD4+ T cell populations depending on their specificity and suggest that the elevated level of immune activation that characterizes chronic HIV infection may be influenced by the persistence of other antigens.
doi:10.1186/1471-2334-13-100
PMCID: PMC3598342  PMID: 23442890
HIV; Immune activation; CD4+ T cells; Antigen persistence; CD38; HLA-DR
8.  CD4 T Cell Responses in Latent and Chronic Viral Infections 
The spectrum of tasks which is fulfilled by CD4 T cells in the setting of viral infections is large, ranging from support of CD8 T cells and humoral immunity to exertion of direct antiviral effector functions. While our knowledge about the differentiation pathways, plasticity, and memory of CD4 T cell responses upon acute infections or immunizations has significantly increased during the past years, much less is still known about CD4 T cell differentiation and their beneficial or pathological functions during persistent viral infections. In this review we summarize current knowledge about the differentiation, direct or indirect antiviral effector functions, and the regulation of virus-specific CD4 T cells in the setting of persistent latent or active chronic viral infections with a particular emphasis on herpes virus infections for the former and chronic lymphocytic choriomeningitis virus infection for the latter.
doi:10.3389/fimmu.2013.00105
PMCID: PMC3651995  PMID: 23717308
CD4 T cells; persistent viral infection; effector functions; differentiation; regulation
9.  Comparison of cytotoxic T lymphocyte efficacy in acute and persistent lymphocytic choriomeningitis virus infection 
Immune responses mediated by cytotoxic T lymphocytes (CTLs) have often been found to be functionally impaired in persistent infections. It is assumed that this impairment contributes to persistence of the infection. In this study, we compare the killing efficacy of CD8+ T-cell responses in mice acutely and persistently infected with the lymphocytic choriomeningitis virus, using an in vivo CTL killing assay. To infer the killing efficacy of CTLs, we developed a new mathematical model describing the disappearance of peptide-pulsed cells from the blood of the mice over time. We estimate a lower half-life for peptide-pulsed cells in acute infection than in persistent infection, which indicates a higher killing efficacy of the CD8+ T-cell response in acute infection. However, by controlling for the different levels of CTLs in acutely and persistently infected mice, we find that CTLs in persistent infection are only two times less efficacious than CTLs in acute infections. These results strongly suggest that the in vivo cytotoxicity of CD8+ T-cell responses in persistent infection is modulated via the number of CTLs rather than their individual functionality.
doi:10.1098/rspb.2011.0453
PMCID: PMC3177638  PMID: 21450739
population biology; within host; cellular immune response; persistent infection; cytotoxic T lymphocyte efficacy; mathematical modelling
10.  Immune Senescence: Relative Contributions of Age and Cytomegalovirus Infection 
PLoS Pathogens  2012;8(8):e1002850.
Immune senescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterised by impaired protective immunity and decreased efficacy of vaccines. Recent clinical, epidemiological and immunological studies suggest that Cytomegalovirus (CMV) infection may be associated with accelerated immune senescence, possibly by restricting the naïve T cell repertoire. However, direct evidence whether and how CMV-infection is implicated in immune senescence is still lacking. In this study, we have investigated whether latent mouse CMV (MCMV) infection with or without thymectomy (Tx) alters antiviral immunity of young and aged mice. After infection with lymphocytic choriomeningitis virus (LCMV) or Vaccinia virus, specific antiviral T cell responses were significantly reduced in old, old MCMV-infected and/or Tx mice compared to young mice. Importantly, control of LCMV replication was more profoundly impaired in aged MCMV-infected mice compared to age-matched MCMV-naïve or young mice. In addition, latent MCMV infection was associated with slightly reduced vaccination efficacy in old Tx mice. In contrast to the prevailing hypothesis of a CMV-mediated restriction of the naïve T cell repertoire, we found similar naïve T cell numbers in MCMV-infected and non-infected mice, whereas ageing and Tx clearly reduced the naïve T cell pool. Instead, MCMV-infection expanded the total CD8+ T cell pool by a massive accumulation of effector memory T cells. Based on these results, we propose a new model of increased competition between CMV-specific memory T cells and any ‘de novo’ immune response in aged individuals. In summary, our results directly demonstrate in a mouse model that latent CMV-infection impairs immunity in old age and propagates immune senescence.
Author Summary
Cytomegalovirus (CMV) persistently infects 50–90% of the human population. After primary infection, constant immune surveillance is required to prevent CMV-related disease. During ageing, increasing T cell resources are expended to keep CMV under control. Recent human studies have suggested that this investment may come at the cost of accelerated immune senescence, a condition describing the age-associated decline of the immune system's functionality. In the present study, we have developed a mouse model to directly investigate whether and how CMV-infection might impair immunity of aged individuals. We demonstrate that old mice with long-lasting CMV-infection are more susceptible to viral infections than old mice without CMV since their virus specific T cell response is suppressed. Contrary to the prevailing hypothesis we found no indication for a CMV-associated shrinking of the naïve T cell compartment. Instead, CMV-infection precipitated a massive expansion of memory T cells. Thus, we propose an alternative mechanism of CMV-enhanced immune senescence based on T cell competition between CMV-specific memory T cells and de novo generated T cell responses. In summary, we provide the first direct evidence that CMV-infection is indeed a propagating factor for poor immunity in the elderly.
doi:10.1371/journal.ppat.1002850
PMCID: PMC3420944  PMID: 22916013
11.  IL-10 Suppression of NK/DC Crosstalk Leads to Poor Priming of MCMV-Specific CD4 T Cells and Prolonged MCMV Persistence 
PLoS Pathogens  2012;8(8):e1002846.
IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10−/− mice led to faster control of lytic viral replication, but this came at the expense of TNF-α mediated immunopathology. Taken together, our data show that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive immune cell crosstalk, thereby impacting beneficially on the ensuing virus-host balance for both the virus and the host.
Author Summary
Cytomegalovirus (CMV) infections are very widespread in mammalian hosts. Despite the fact that CMVs are usually well controlled by the immune system, they cause persistent life-long infection and have evolved a number of strategies to effectively modulate or hide from host immunity. Since the establishment of an immunosuppressive environment favors virus persistence, IL-10 is one of the host targets that CMVs actively use to tune the virus-host balance toward their own benefit, resulting in prolonged virus persistence and hence increased chance for horizontal transmission. Here, we delineate the mechanisms of how IL-10 exerts its powerful immune-suppressing function in the context of murine cytomegalovirus (MCMV) infection. We found that IL-10 specifically restrains the priming of MCMV-specific CD4 T cell responses by suppressing dendritic cell (DC) - natural killer cell (NK) crosstalk during acute MCMV infection. Target molecules mediating this bi-directional crosstalk between DCs and NK cells were the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 and all of them were markedly suppressed by IL-10. A consequence resulting from this impeded DC-NK cross-talk by IL-10, leading to poor priming of MCMV-specific CD4 T cell responses was increased lytic CMV persistence and reduced development of host tissue damage. Our study indicates that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive crosstalk, thereby imparting on the ensuing virus-host balance for the benefit of both the virus and the host.
doi:10.1371/journal.ppat.1002846
PMCID: PMC3410900  PMID: 22876184
12.  T Cell Memory in the Context of Persistent Herpes Viral Infections 
Viruses  2012;4(7):1116-1143.
The generation of a functional memory T cell pool upon primary encounter with an infectious pathogen is, in combination with humoral immunity, an essential process to confer protective immunity against reencounters with the same pathogen. A prerequisite for the generation and maintenance of long-lived memory T cells is the clearance of antigen after infection, which is fulfilled upon resolution of acute viral infections. Memory T cells play also a fundamental role during persistent viral infections by contributing to relative control and immuosurveillance of active replication or viral reactivation, respectively. However, the dynamics, the phenotype, the mechanisms of maintenance and the functionality of memory T cells which develop upon acute/resolved infection as opposed to chronic/latent infection differ substantially. In this review we summarize current knowledge about memory CD8 T cell responses elicited during α-, β-, and γ-herpes viral infections with major emphasis on the induction, maintenance and function of virus-specific memory CD8 T cells during viral latency and we discuss how the peculiar features of these memory CD8 T cell responses are related to the biology of these persistently infecting viruses.
doi:10.3390/v4071116
PMCID: PMC3407898  PMID: 22852044
herpes virus infection; CD8 T cells; memory inflation: secondary lymphoid organs; peripheral tissues; CD8 T cell function; CD8 T cell maintenance; CD8 T cell phenotype
13.  Antigen-Dependent and -Independent Mechanisms of T and B Cell Hyperactivation during Chronic HIV-1 Infection▿ 
Journal of Virology  2011;85(23):12102-12113.
Continuous loss of CD4+ T lymphocytes and systemic immune activation are hallmarks of untreated chronic HIV-1 infection. Chronic immune activation during HIV-1 infection is characterized by increased expression of activation markers on T cells, elevated levels of proinflammatory cytokines, and B cell hyperactivation together with hypergammaglobulinemia. Importantly, hyperactivation of T cells is one of the best predictive markers for progression toward AIDS, and it is closely linked to CD4+ T cell depletion and sustained viral replication. Aberrant activation of T cells is observed mainly for memory CD4+ and CD8+ T cells and is documented, in addition to increased expression of surface activation markers, by increased cell cycling and apoptosis. Notably, the majority of these activated T cells are neither HIV specific nor HIV infected, and the antigen specificities of hyperactivated T cells are largely unknown, as are the exact mechanisms driving their activation. B cells are also severely affected by HIV-1 infection, which is manifested by major changes in B cell subpopulations, B cell hyperactivation, and hypergammaglobulinemia. Similar to those of T cells, the mechanisms underlying this aberrant B cell activation remain largely unknown. In this review, we summarized current knowledge about proposed antigen-dependent and -independent mechanisms leading to lymphocyte hyperactivation in the context of HIV-1 infection.
doi:10.1128/JVI.05607-11
PMCID: PMC3209369  PMID: 21849433
14.  Non-Hematopoietic Cells in Lymph Nodes Drive Memory CD8 T Cell Inflation during Murine Cytomegalovirus Infection 
PLoS Pathogens  2011;7(10):e1002313.
During human and murine cytomegalovirus (MCMV) infection an exceptionally large virus-specific CD8 T cell pool is maintained in the periphery lifelong. This anomalous response is only seen for specific subsets of MCMV-specific CD8 T cells which are referred to as 'inflationary T cells'. How memory CD8 T cell inflation is induced and maintained is unclear, though their activated phenotype strongly suggests an involvement of persistent antigen encounter during MCMV latency. To dissect the cellular and molecular requirements for memory CD8 T cell inflation, we have generated a transgenic mouse expressing an MHC class I-restricted T cell receptor specific for an immunodominant inflationary epitope of MCMV. Through a series of adoptive transfer experiments we found that memory inflation was completely dependent on antigen presentation by non-hematopoietic cells, which are also the predominant site of MCMV latency. In particular, non-hematopoietic cells selectively induced robust proliferation of inflationary CD8 T cells in lymph nodes, where a majority of the inflationary CD8 T cells exhibit a central-memory phenotype, but not in peripheral tissues, where terminally differentiated inflationary T cells accumulate. These results indicate that continuous restimulation of central memory CD8 T cells in the lymph nodes by infected non-hematopoietic cells ensures the maintenance of a functional effector CD8 T pool in the periphery, providing protection against viral reactivation events.
Author Summary
Cytomegaloviruses (CMVs) infect the majority of the human population and persist lifelong via latency. CMV latency is thought to be a dynamic state, characterized by stochastic viral reactivation events coupled to CMV-derived antigen presentation. In support of this hypothesis is the exceptionally large CMV-specific CD8 T cell response which constitutes an integral part of immune surveillance of CMV reactivation. Conversely, it may also contribute to immune senescence as it significantly shapes the overall CD8 T cell pool in bias of CMV-specificity. In mice, only a subset of CMV-specific CD8 T cells, also called ‘inflationary CD8 T cells’, contribute to this large response. The mechanism leading to the selective accumulation and persistence of memory CD8 T cells during MCMV latency is largely unknown. Here, we unraveled the mechanisms of memory CD8 T cell inflation using a newly generated TCR transgenic mouse with specificity for an immunodominant inflationary MCMV epitope. We show that antigen presentation on non-hematopoietic cells is essential for memory inflation and that memory inflation in peripheral tissues is fueled by lymph node-resident central memory CD8 T cells, being locally reactivated by non-hematopoietic cells, inducing their local expansion and migration to peripheral tissues where they control viral reactivation events.
doi:10.1371/journal.ppat.1002313
PMCID: PMC3203160  PMID: 22046127
15.  Absence of Cross-Presenting Cells in the Salivary Gland and Viral Immune Evasion Confine Cytomegalovirus Immune Control to Effector CD4 T Cells 
PLoS Pathogens  2011;7(8):e1002214.
Horizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control in secretory organs. CD4 T cells were crucial to cease MCMV replication in the salivary gland (SG) via direct secretion of IFNγ that initiated antiviral signaling on non-hematopoietic cells. In contrast, CD4 T cell helper functions for CD8 T cells or B cells were dispensable. Despite SG-resident MCMV-specific CD8 T cells being able to produce IFNγ, the absence of MHC class I molecules on infected acinar glandular epithelial cells due to viral immune evasion, and the paucity of cross-presenting antigen presenting cells (APCs) prevented their local activation. Thus, local activation of MCMV-specific T cells is confined to the CD4 subset due to exclusive presentation of MCMV-derived antigens by MHC class II molecules on bystander APCs, resulting in IFNγ secretion interfering with viral replication in cells of non-hematopoietic origin.
Author Summary
Cytomegaloviruses (CMVs) infect 50 to 90 % of the world's population and cause severe clinical complication in immunosuppressed individuals. An important tissue for horizontal transmission is the salivary gland (SG). CD4 T cells are crucial for viral control in this organ. However, how CD4 T cells control MCMV and why CD8 T cells, important effector cells in other organs, are inefficient in the SG, remains unclear. Here we show that CD4 T cells exert direct antiviral effector rather than helper functions by secretion of IFNγ acting on non-hematopoietic cells. Although SG-resident CD8 T cells were able to produce IFNγ and outnumbered CD4 T cells, absence of MHC class I expression on infected cells due to CMV-encoded immune evasion genes and concomitant absence of cross-presenting antigen presenting cells prohibited antigen recognition by CD8 T cells. Deletion of CMV-encoded immune evasion genes enabled CD8 T cells to control MCMV replication in the SG in absence of CD4 T cells. Hence, CMV control depends on direct antiviral functions of CD4 T cells because of exclusive MHC class II-restricted CMV antigen presentation by bystander APCs in the SG, exemplifying a strategy of effective immune evasion by which CMVs to promote their own transmission.
doi:10.1371/journal.ppat.1002214
PMCID: PMC3161985  PMID: 21901102
16.  HIV-1 replication activates CD4+ T cells with specificities for persistent herpes viruses 
EMBO Molecular Medicine  2010;2(6):231-244.
Hyperactivation of CD4+ T cells is a hallmark of untreated HIV-1 infection. The antigenic specificities of activated CD4+ T cells and the underlying mechanisms leading to their activation remain thus far elusive. We report here that during HIV rebound the dynamics of HIV-specific CD4+ T cells is highly correlated with the dynamics of CD4+ T cells specific for persistent antigens derived from various members of the herpes virus family, whereas CD4 responses towards non-persistent antigens were unaffected by HIV replication. Notably, the dynamics of HIV and herpes viral antigen-specific CD4+ T cells responses correlated with the expression level of activation markers on dendritic cells (DCs) and activated DCs were more potent in restimulating memory T cells. These data strongly suggest that HIV replication costimulates activation of CD4+ T cells specific for persistent herpes viral antigens via activation of DCs. We propose that a large proportion of activated T cells during untreated HIV infection may be specific for herpes viral antigens and identify a novel mechanism contributing to chronic immune activation in untreated HIV-1 infection.
doi:10.1002/emmm.201000075
PMCID: PMC3377320  PMID: 20533427
CD4+ T cells; chronic immune activation; dendritic cells; herpes viruses; HIV pathogenesis
17.  No evidence for competition between cytotoxic T-lymphocyte responses in HIV-1 infection 
Strong competition between cytotoxic T-lymphocytes (CTLs) specific for different epitopes in human immunodeficiency virus (HIV) infection would have important implications for the design of an HIV vaccine. To investigate evidence for this type of competition, we analysed CTL response data from 97 patients with chronic HIV infection who were frequently sampled for up to 96 weeks. For each sample, CTL responses directed against a range of known epitopes in gag, pol and nef were measured using an enzyme-linked immunospot assay. The Lotka–Volterra model of competition was used to predict patterns that would be expected from these data if competitive interactions materially affect CTL numbers. In this application, the model predicts that when hosts make responses to a larger number of epitopes, they would have diminished responses to each epitope and that if one epitope-specific response becomes dramatically smaller, others would increase in size to compensate; conversely if one response grows, others would shrink. Analysis of the experimental data reveals results that are wholly inconsistent with these predictions. In hosts who respond to more epitopes, the average epitope-specific response tends to be larger, not smaller. Furthermore, responses to different epitopes almost always increase in unison or decrease in unison. Our findings are therefore inconsistent with the hypothesis that there is competition between CTL responses directed against different epitopes in HIV infection. This suggests that vaccines that elicit broad responses would be favourable because they would direct a larger total response against the virus, in addition to being more robust to the effects of CTL escape.
doi:10.1098/rspb.2009.1232
PMCID: PMC2817101  PMID: 19776069
cytotoxic T-lymphocytes; human immunodeficiency virus; competition; mathematical model
18.  Functional and biophysical characterization of an HLA-A*6801-restricted HIV-specific T cell receptor 
European Journal of Immunology  2007;37(2):479-486.
HLA-A*6801 exhibits several unusual features. First, it is known to bind weakly to CD8 due to the presence of an A245V substitution in the α3 domain. Second, it is able to accommodate unusually long peptides as a result of peptide ‘kinking’ in the binding groove. Third, CD8+ cytotoxic T lymphocytes that recognise HLA-A*6801-restricted antigens can tolerate substantial changes in the peptide sequence without apparent loss of recognition. In addition, it has been suggested that HLA-A68-restricted TCR might bind with higher affinity than other TCR due to their selection in the presence of a decreased contribution from CD8. Here we (1) examine monoclonal T cell recognition of an HLA-A*6801-restricted HIV-1 Tat-derived 11-amino acid peptide (ITKGLGISYGR) and natural variant sequences thereof; (2) measure the affinity and kinetics of a TCR/pHLA-A68 interaction biophysically for the first time, showing that equilibrium binding occurs within the range previously determined for non-HLA-A68-restricted TCR (KD approx. 7 μM); and (3) show that “normalization” of the non-canonical HLA-A*6801 CD8-binding domain enhances recognition of agonist peptides without inducing non-specific activation. This latter effect may provide a fundamental new mechanism with which to enhance T cell immunity to specific antigens.
doi:10.1002/eji.200636243
PMCID: PMC2699040  PMID: 17273992
HIV-1; HLA-A68; TCR
19.  Emergence of Polyfunctional CD8+ T Cells after Prolonged Suppression of Human Immunodeficiency Virus Replication by Antiretroviral Therapy▿  
Journal of Virology  2008;82(7):3391-3404.
Progressive human immunodeficiency virus type 1 (HIV-1) infection is often associated with high plasma virus load (pVL) and impaired CD8+ T-cell function; in contrast, CD8+ T cells remain polyfunctional in long-term nonprogressors. However, it is still unclear whether CD8+ T-cell dysfunction is the cause or the consequence of high pVLs. Here, we conducted a longitudinal functional and phenotypic analysis of virus-specific CD8+ T cells in a cohort of patients with chronic HIV-1 infection. During the initiation and maintenance of successful antiretroviral therapy (ART), we assessed whether the level of pVL was associated with the degree of CD8+ T-cell dysfunction. Under viremic conditions, HIV-specific CD8+ T cells were dysfunctional with respect to cytokine secretion (gamma interferon, interleukin-2 [IL-2], and tumor necrosis factor alpha), and their phenotype suggested limited potential for proliferation. During ART, cytokine secretion by HIV-specific CD8+ T cells was gradually restored, IL-7Rα and CD28 expression increased dramatically, and PD-1 levels declined. Thus, prolonged ART-induced reduction of viral replication and, hence, presumably antigen exposure in vivo, allows a significant functional restoration of CD8+ T cells with the appearance of polyfunctional cells. These findings indicate that the level of pVL as a surrogate for antigen load has a dominant influence on the phenotypic and functional profile of virus-specific CD8+ T cells.
doi:10.1128/JVI.02383-07
PMCID: PMC2268491  PMID: 18199637
20.  Peroxiredoxin 6 is required for blood vessel integrity in wounded skin 
The Journal of Cell Biology  2007;179(4):747-760.
Peroxiredoxin 6 (Prdx6) is a cytoprotective enzyme with largely unknown in vivo functions. Here, we use Prdx6 knockout mice to determine its role in UV protection and wound healing. UV-mediated keratinocyte apoptosis is enhanced in Prdx6-deficient mice. Upon skin injury, we observe a severe hemorrhage in the granulation tissue of knockout animals, which correlates with the extent of oxidative stress. At the ultrastructural level endothelial cells appear highly damaged, and their rate of apoptosis is enhanced. Knock-down of Prdx6 in cultured endothelial cells also increases their susceptibility to oxidative stress, thus confirming the sensitivity of this cell type to loss of Prdx6. Wound healing studies in bone marrow chimeric mice demonstrate that Prdx6-deficient inflammatory and endothelial cells contribute to the hemorrhage phenotype. These results provide insight into the cross-talk between hematopoietic and resident cells at the wound site and the role of reactive oxygen species in this interplay.
doi:10.1083/jcb.200706090
PMCID: PMC2080929  PMID: 18025307
22.  O Mannosylation of α-Dystroglycan Is Essential for Lymphocytic Choriomeningitis Virus Receptor Function 
Journal of Virology  2005;79(22):14297-14308.
α-Dystroglycan (α-DG) was identified as a common receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses including the human pathogenic Lassa fever virus. Initial work postulated that interactions between arenavirus glycoproteins and α-DG are based on protein-protein interactions. We found, however, that susceptibility toward LCMV infection differed in various cell lines despite them expressing comparable levels of DG, suggesting that posttranslational modifications of α-DG would be involved in viral receptor function. Here, we demonstrate that glycosylation of α-DG, and in particular, O mannosylation, which is a rare type of O-linked glycosylation in mammals, is essential for LCMV receptor function. Cells that are defective in components of the O-mannosylation pathway showed strikingly reduced LCMV infectibility. As defective O mannosylation is associated with severe clinical symptoms in mammals such as congenital muscular dystrophies, it is likely that LCMV and potentially other arenaviruses may have selected this conserved and crucial posttranslational modification as the primary target structure for cell entry and infection.
doi:10.1128/JVI.79.22.14297-14308.2005
PMCID: PMC1280192  PMID: 16254364
23.  Low Human Immunodeficiency Virus Envelope Diversity Correlates with Low In Vitro Replication Capacity and Predicts Spontaneous Control of Plasma Viremia after Treatment Interruptions 
Journal of Virology  2005;79(14):9026-9037.
Genetic diversity of viral isolates in human immunodeficiency virus (HIV)-infected individuals varies substantially. However, it remains unclear whether HIV-related disease progresses more rapidly in patients harboring virus swarms with low or high diversity and, in the same context, whether high or low diversity is required to induce potent humoral and cellular immune responses. To explore whether viral diversity predicts virologic control, we studied HIV-infected patients who received antiretroviral therapy (ART) for years before undergoing structured treatment interruptions (STI). Viral diversity before initiation of ART and the ability of the patients to contain viremia after STI and final cessation of treatment was evaluated. Seven out of 21 patients contained plasma viremia at low levels after the final treatment cessation. Clonal sequences encompassing the envelope C2V3C3 domain derived from plasma prior to treatment, exhibited significantly lower diversity in these patients compared to those derived from patients with poor control of viremia. Viral diversity pre-ART correlated with the viral replication capacity of rebounding virus isolates during STI. Neutralizing antibody activity against autologous virus was significantly higher in patients who controlled viremia and was associated with lower pretreatment diversity. No such association was found with binding antibodies directed to gp120. In summary, lower pretreatment viral diversity was associated with spontaneous control of viremia, reduced viral replication capacity and higher neutralizing antibody titers, suggesting a link between viral diversity, replication capacity, and neutralizing antibody activity.
doi:10.1128/JVI.79.14.9026-9037.2005
PMCID: PMC1168724  PMID: 15994796
24.  HIV-1–specific CD4+ T lymphocyte turnover and activation increase upon viral rebound 
Journal of Clinical Investigation  2005;115(2):443-450.
HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1–specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned.
doi:10.1172/JCI200523084
PMCID: PMC544605  PMID: 15668739
25.  Immediate Cytotoxicity But Not Degranulation Distinguishes Effector and Memory Subsets of CD8+ T Cells 
CD8+ T cells play a central role in the resolution and containment of viral infections. A key effector function of CD8+ T cells is their cytolytic activity toward infected cells. Here, we studied the regulation of cytolytic activity in naive, effector, and central versus effector memory CD8+ T cells specific for the same glycoprotein-derived epitope of lymphocytic choriomeningitis virus. Our results show that the kinetics of degranulation, assessed by a novel flow cytometric based assay, were identical in effector and both subsets of memory CD8+ T cells, but absent in naive CD8+ T cells. However, immediate cytolytic activity was most pronounced in effector T cells, low in effector memory T cells, and absent in central memory T cells, correlating with the respective levels of cytolytic effector molecules present in lytic granules. These results indicate that an inherent program of degranulation is a feature of antigen-experienced cells as opposed to naive CD8+ T cells and that the ability of CD8+ T cells to induce target cell apoptosis/death is dependent on granule protein content rather than on the act of degranulation itself. Furthermore, these results provide a potential mechanism by which central memory CD8+ T cell–mediated death of antigen-presenting cells within the lymph node is avoided.
doi:10.1084/jem.20031799
PMCID: PMC2211884  PMID: 15051762
cytotoxic T cell; central memory; effector memory; lytic granules; virus

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